A selected lipid type virus, viral hepatitis, has been recognized as an important and serious sequela of parenteral exposure to blood and blood components since the early 1940s. It was originally believed that all such blood-associated hepatitis was caused by the serum hepatitis virus (now called the hepatitis B virus, or HBV). Subsequently, the development of sensitive assays for infection with this virus revealed that only approximately 1/3 of transfusion-associated hepatitis was caused by the HBV. It was thought that the remaining hepatitis was caused by the hepatitis A virus. However, the development of sensitive assays for HAV led to the recognition of a new hepatitis virus, the non-A, non-B hepatitis virus (NANB) in 1975. The successful application of sensitive screening tests for HBV to blood donors has resulted in a decrease (but not disappearance) of HBV in transfusion-associated hepatitis; at present approximately 90 percent of such hepatitis is caused by non-A, non-B agents.
Similarly, hepatitis following administration of blood products such a antihemophilic factor was thought to be caused solely by HBV. However, in the late 1970s, the association of NANB agents with administration of antihemophilic factor to hemophiliacs was reported and confirmed. As the transfusion-associated hepatitis, the application of serologic screening methods to plasma donors has resulted in a relative decrease in the importance of HBV in such blood product-associated hepatitis.
Non-A, Non-B hepatitis is the major cause of transfusion associated hepatitis in the United States. Presently less than 10% of post-transfusion cases are caused by the hepatitis B virus. Of the remainder, cytomegalovirus may account for a small proportion but the vast majority are caused by an as yet unidentified agent. There is a large amount of evidence supporting a transmissable agent as the cause of NANBH. This includes transmission studies done in both humans and non-human primates. Chimpanzees and marmoset monkeys have both been shown to be susceptible to infection by at least some NANBH agents. Though very costly and cumbersome to work with, these animals can be used to aid in the characterization of the infectious agent of NANBH.
Unfortunately, serologic tests for the detection of NANB agents are not available for detection of potentially infectious donors because the agents have not been adequately identified and characterized despite extensive efforts to do so. Therefore, blood and blood products remain potential sources for transmission of hepatitis agents to recipients. The resultant hepatitis can be quite serious, even life-threatening, and can result in not only acute hepatitis but also chronic hepatitis in a significant proportion of cases.
For these reasons attempts to inactivate hepatitis agents in blood and blood products have been pursued with vigor. Such approaches have included the use of heat, the addition of anti-HBV antibody, the use of solid immunoadsorbents or other chemical-specific adsorbents, exposure to ultraviolet radiation, the addition of certain inactivating substances, such as beta-propriolactone, surface-active substances, etc. None of the approaches has been entirely successful and some have introduced an added potential risk (e.g., beta-propiolactone is carcinogenic). Failure of these approaches stems from relative resistance of the agents to physical or chemical inactivation, particularly when in the presence of high protein concentrations as occurs with blood products and from limited knowledge about the nature of the hepatitis agents, especially the NANB agents.
As part of a systematic characterization of NANB agents by standardized virologic methods, the present inventors first established that HBV and at least one NANB agent contain lipids essential for the integrity and viability of the viruses. This was established by exposing the viruses to a potent lipid solvent (chloroform) and demonstrating that such chloroform-extracted viruses were rendered non-infectious in a suitable susceptible host, the chimpanzee (Pan troglodytes).
It is understood that the present invention applies to all lipid containing NANBH particles which of necessity may be more than one particle as, for example, the more recently discovered delta particle. In exploring other viruses, the inventors also found that any virus which contains an essential lipid can be inactivated by lipid solvents. Thus, this invention expands the parent invention in two significant ways: (1) the class of viruses affected was expanded; and (2) the class of effective lipid solvents applicable in this procedure was expanded.
The present invention relates to a method of inactivating a lipid virus in a protein carrier where said lipid virus is defined to include members of the herpesvirus group (cytomegalovirus, Epstein-Barr virus, herpes zoster virus, herpesvirus type 1 and herpesvirus type 2), the delta agent (a type of non-A, non-B hepatitis virus), and members of other blood-borne virus groups including the togaviruses (including rubella virus) and the bunyaviruses, retroviruses (including the human T-cell leukemia virus), orthomyxoviruses (influenza), paramyxoviruses (measles, mumps), rhabdoviruses (rabies, Marburg agent), and arenaviruses (Lassa fever, other hemorrhagic fevers) as well as other members of the poxvirus group such as vaccinia virus. Other viruses, known or suspected putative agent of acquired immune deficiency syndrome, AIDS, are included as viruses containing essential lipids.
Inactivation is achieved by contacting the virus (in a carrier) for an abbreviated time period at generally ambient temperatures, with a preferred halohydrocarbon treating agent.
This treating agent or solvent is defined to include at least the following: chloroform and chloroform/alkanol mixtures, preferred; dialkyl ethers such as ethyl ether; lower alcohols such as ethanol, methanol, and butanol; and the fluorocarbons (trichlorotrifluoroethane) which include the most common Freon.RTM. or Genetron.RTM. agents such as CCl.sub.3 F, CH.sub.2 F.sub.2, CCl.sub.2 F.sub.2, CCl.sub.2 FCCLF.sub.2. Freons are defined as chlorofluoro carbon of one or two carbon atoms. These may be fluorinated derivatives of methane and ethane. Additional agents are alkylene halides such as dichloroethylene. These treating agents are administered in an amount of approximately 5-50% v/v. The treating agent or solvent may also consist of mixtures of any of the above, with chloroform/lower alkanol being preferred. Although chloroform was used as the inactivating agent to demonstrate the principle of inactivation of hepatitis viruses with a lipid solvent, in practice, these other lipid solvents substitute for chloroform and are effective in inactivating virtually all lipid-containing viruses with the exception of the poxviruses. Thus, the concept of inactivating lipid-containing viruses by extracting their essential lipids with a lipid solvent does not require the preferred chloroform or chloroform/alkanol mixtures (such as chloroform/methanol in a 1:4 to 4:1 ratio), but can be performed with selected other lipid solvents selected on the basis of other characteristics (e.g., lack of toxicity, ease of handling, cost, etc.).
The period of time for treatment is about 10 minutes to five hours and the generally ambient temperature is from about 4.degree. C. to 40.degree. C. The five-hour upper limit may be expandable to any reasonable amount of time since it is the lower range period that determines the efficacy of the process. The treating agent of choice is chloroform, CHCl.sub.3, or CHCl.sub.3 +a lower alkanol (i.e., methanol) and can be used in a method utilizing both extraction of the aqueous blood products by physical means (e.g., centrifugation), or by extraction of the lyophilized dried blood products and the removal of the solvent by other appropriate means such as vacuum evaporation. Generally, these two processes can be distinguished in that in the latter there is a reduction to powder form before treatment, a process which requires greater protection of the products that have been lyophilized.
The process of this invention may require extraction of the virus in dry powder form. However, the virus in its liquid state is preferred. (See Example 2).
The inactivation of HBV and NANBV has been shown in animals such as chimpanzees.
Diethyl ether is an effective inactivating agent for destroying endotoxin or infectivity of non-hepatitis viruses and preventing clotting activation. However, it is a less efficient solvent of lipids. Also, the use of diethyl ether is not recommended for the broader use of the present agents in the nuclization of pox virus; such pox virus may be illustrated by smallpox and vaccinia virus. Not only is diethyl ether noted but other ethers, such as phenoxy, polyethoxy ethanol, and compounds of the general formula RC.sub.6 H.sub.4 (OC.sub.2 H.sub.4).sub.n OH are also noted.
The present use of chloroform and the Freon or Genetron fluorocarbon agents has been found useful in so-called lipid virus which include HBV and NANBH and also include those virus particles which have a lipid outer coat which is suceptible to removal by treatment.